Kinases known to be associated with tumorigenesis include the Raf serine/threonine kinases and the receptor tyrosine kinases (RTKs).
The Raf serine/threonine kinases are essential components of the Ras/Mitogen-Activated Protein Kinase (MAPK) signaling module that controls a complex transcriptional program in response to external cellular stimuli. Raf genes code for highly conserved serine-threonine-specific protein kinases which are known to bind to the ras oncogene. They are part of a signal transduction pathway believed to consist of receptor tyrosine kinases, p21 ras, Raf protein kinases, Mek1 (ERK activator or MAPKK) kinases and ERK (MAPK) kinases, which ultimately phosphorylate transcription factors. In this pathway Raf kinases are activated by Ras and phosphorylate and activate two isoforms of Mitogen-Activated Protein Kinase Kinase (called Mek1 and Mek2), that are dual specificity threonine/tyrosine kinases. Both Mek isoforms activate Mitogen Activated Kinases 1 and 2 (MAPK, also called Extracellular Ligand Regulated Kinase 1 and 2 or Erk1 and Erk2). The MAPKs phosphorylate many substrates including transcription factors and in so doing set up their transcriptional program. Raf kinase participation in the Ras/MAPK pathway influences and regulates many cellular functions such as proliferation, differentiation, survival, oncogenic transformation and apoptosis.
Both the essential role and the position of Raf in many signaling pathways have been demonstrated from studies using deregulated and dominant inhibitory Raf mutants in mammalian cells as well as from studies employing biochemical and genetic techniques of model organisms. In many cases, the activation of Raf by receptors that stimulate cellular tyrosine phosphorylation is dependent on the activity of Ras, indicating that Ras functions upstream of Raf. Upon activation, Raf-1 then phosphorylates and activates Mek1, resulting in the propagation of the signal to downstream effectors, such as MAPK (mitogen-activated protein kinase; Crews et al., 1993, Cell 74:215). The Raf serine/threonine kinases are considered to be the primary Ras effectors involved in the proliferation of animal cells (Avruch et al., 1994, Trends Biochem. Sci. 19:279).
Raf kinase has three distinct isoforms, Raf-1 (c-Raf), A-Raf, and B-Raf, distinguished by their ability to interact with Ras, to activate MAPK kinase pathway, tissue distribution and sub-cellular localization (Marias et al., Biochem. J. 351:289-305, 2000; Weber et al., Oncogene 19:169-176, 2000; Pritchard et al., Mol. Cell. Biol. 15:6430-6442, 1995). Activating mutation of one of the Ras genes can be seen in about 20% of all tumors and the Ras/Raf/MEK/ERK pathway is activated in about 30% of all tumors (Bos et al., Cancer Res. 49:4682-4689, 1989; Hoshino et al., Oncogene 18:813-822, 1999). Recent studies have shown that B-Raf mutation in the skin nevi is a critical step in the initiation of melanocytic neoplasia (Pollock et al., Nature Genetics 25: 1-2, 2002). Furthermore, most recent studies have disclosed that activating mutation in the kinase domain of B-Raf occurs in about 66% of melanomas, 12% of colon carcinoma and 14% of liver cancer (Davies et al., Nature 417:949-954, 2002; Yuen et al., Cancer Research 62:6451-6455, 2002; Brose et al., Cancer Research 62:6997-7000, 2002).
Melanoma, which continues to represent a significant unmet medical need, is a complex multigenic disease with a poor prognosis, especially in the advanced metastatic state. Activating somatic mutations in the B-Raf proto-oncogene have recently been discovered in a variety of malignancies, and most frequently in melanoma. Approximately 70% of melanoma express a mutated and activated form of B-Raf (V600E), making it an excellent target for drug development. Furthermore, another 10-15% of melanomas express mutant N-Ras, further demonstrating the importance of the MAPK pathway in the growth and survival of melanoma cells.
Inhibitors of the Ras/Raf/MEK/ERK pathway at the level of Raf kinases can potentially be effective as therapeutic agents against tumors with over-expressed or mutated receptor tyrosine kinases, activated intracellular tyrosine kinases, tumors with aberrantly expressed Grb2 (an adapter protein that allows stimulation of Ras by the Sos exchange factor) as well as tumors harboring activating mutations of Raf itself. In the early clinical trials inhibitors of Raf-1 kinase that also inhibit B-Raf have shown promise as therapeutic agents in cancer therapy (Crump, Current Pharmaceutical Design 8:2243-2248, 2002; Sebastien et al., Current Pharmaceutical Design 8: 2249-2253, 2002).
Disruption of Raf expression in cell lines through the application of RNA antisense technology has been shown to suppress both Ras and Raf-mediated tumorigenicity (Kolch et al., Nature 349:416-428, 1991; Monia et al., Nature Medicine 2(6):668-675, 1996). It has also been shown that the administration of deactivating antibodies against Raf kinase or the co-expression of dominant negative Raf kinase or dominant negative MEK, the substrate of Raf kinase, leads to the reversion of transformed cells to the normal growth phenotype (see Daum et al., Trends Biochem. Sci 1994, 19:474-80; Fridman et al. J. Biol. Chem. 1994, 269:30105-8).
Several Raf kinase inhibitors have been described as exhibiting efficacy in inhibiting-tumor cell proliferation in vitro and/or in vivo assays (see, e.g., U.S. Pat. Nos. 6,391,636, 6,358,932, 6,037,136, 5,717,100, 6,458,813, 6,204,467, and 6,268,391). Other patents and patent applications suggest the use of Raf kinase inhibitors for treating leukemia (see, e.g., U.S. Pat. Nos. 6,268,391, and 6,204,467, and published U.S. Patent Application Nos. 20020137774; 20020082192; 20010016194; and 20010006975), or for treating breast cancer (see, e.g., U.S. Pat. Nos. 6,358,932, 5,717,100, 6,458,813, 6,268,391, and 6,204,467, and published U.S. Patent Application No. 20010014679).
Angiogenesis also plays an important role in the growth of cancer cells. It is known that once a nest of cancer cells reaches a certain size, roughly 1 to 2 mm in diameter, the cancer cells must develop a blood supply in order for the tumor to grow larger as diffusion will not be sufficient to supply the cancer cells with enough oxygen and nutrients. Thus, inhibition of angiogenesis is expected to inhibit the growth of cancer cells.
Receptor tyrosine kinases (RTKs) are transmembrane polypeptides that regulate developmental cell growth and differentiation, remodeling and regeneration of adult tissues (Mustonen, T. et al., J. Cell Biology 129:895-898, 1995; van der Geer, P. et al., Ann Rev. Cell Biol. 10:251-337, 1994). Polypeptide ligands, known as growth factors or cytokines, are known to activate RTKs. Signaling RTKs involves ligand binding and a shift in conformation in the external domain of the receptor resulting in its dimerization (Lymboussaki, A. “Vascular Endothelial Growth Factors and their Receptors in Embryos, Adults, and in Tumors” Academic Dissertation, University of Helsinki, Molecular/Cancer Biology Laboratory and Department of Pathology, Haartman Institute, 1999; Ullrich, A. et al., Cell 61:203-212, 1990). Binding of the ligand to the RTK results in receptor trans-phosphorylation at specific tyrosine residues and subsequent activation of the catalytic domains for the phosphorylation of cytoplasmic substrates (Id).
Two subfamilies of RTKs are specific to the vascular endothelium. These include the vascular endothelial growth factor (VEGF) subfamily and the Tie receptor subfamily. Class V RTKs include VEGFR1 (FLT-1), VEGFR2 (KDR (human), Flk-1 (mouse)), and VEGFR3 (FLT-4) (Shibuya, M. et al., Oncogene 5:519-525, 1990; Terman, B. et al., Oncogene 6:1677-1683, 1991; Aprelikova, O. et al., Cancer Res. 52:746-748, 1992). Members of the VEGF subfamily have been described as being able to induce vascular permeability and endothelial cell proliferation and further identified as a major inducer of angiogenesis and vasculogenesis (Ferrara, N. et al., Endocrinol. Rev. 18:4-25, 1997).
VEGF is known to specifically bind to RTKs including FLT-1 and Flk-1 (DeVries, C. et al., Science 255:989-991, 1992; Quinn, T. et al., Proc. Natl. Acad. Sci. 90:7533-7537, 1993). VEGF stimulates the migration and proliferation of endothelial cells and induces angiogenesis both in vitro and in vivo (Connolly, D. et al., J. Biol. Chem. 264:20017-20024, 1989; Connolly, D. et al., J. Clin. Invest. 84:1470-1478, 1989; Ferrara, N. et al., Endocrinol. Rev. 18:4-25, 1997; Leung, D. et al., Science 246:1306-1309, 1989; Plouet, J. et al., EMBO J 8:3801-3806, 1989).
Studies in various cultured endothelial cell systems have established that VEGFR2 mediates the majority of downstream effects of VEGF in angiogenesis (Wey S. et al., Clinical Advances in Hematology and Oncology, 2:37-45, 2004). VEGFR2 mediated proliferation of endothelial cells is believed to involve activation of the Ras/Raf/Mek/Erk pathway (Veikkola T. et al., Cancer Res 60:203-212, 2000). VEGFR2 expression has been observed in melanoma, breast cancer, bladder cancer, lung cancer, thyroid cancer, prostate cancer, and ovarian cancer (see Wey et al., supra). Neutralizing monoclonal antibodies to VEGFR2 (KDR) have been shown to be efficacious in blocking tumor angiogenesis (see Kim et al., Nature 362:841, 1993; Rockwell et al., Mol. Cell. Differ. 3:315, 1995). Because angiogenesis is known to be critical to the growth of cancer and to be controlled by VEGF and VEGF-RTK, substantial efforts have been undertaken to develop compounds which inhibit or retard angiogenesis and inhibit VEGF-RTK.
Platelet derived growth factor receptor kinase (PDGFR) is another type of RTK. PDGF expression has been shown in a number of different solid tumors, from glioblastomas and osteosarcoma to prostate carcinomas. In these various tumor types, the biological role of PDGF signaling can vary from autocrine stimulation of cancer cell growth to more subtle paracrine interactions involving adjacent stroma and angiogenesis. PDGF interacts with tyrosine kinases receptors PDGFRα and PDGFRβ. Therefore, inhibiting the PDGFR kinase activity with small molecules is expected to interfere with tumor growth and angiogenesis.
The fibroblast growth factor receptor kinases (FGFRs) represent another type of RTKs. The fibroblast growth factors are a family of polypeptide growth factors involved in a variety of activities, including mitogenesis, angiogenesis, and wound healing. They comprise a family of related but individually distinct tyrosine kinase receptors containing an extracellular domain with either 2 or 3 immunoglobulin (Ig)-like domains, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. The fibroblast growth factor receptors that have been identified include FGFR1 (Ruta, M et al, Oncogene 3:9-15, 1988); FGFR2 (Dionne, C et al., Cytogenet. Cell Genet. 60:34-36, 1992); FGFR3 (Keegan, K et al., Proc. Nat. Acad. Sci. 88:1095-1099, 1991); and FGFR4 (Partanen, J et al., EMBO J. 10:1347-1354, 1991).
The role of the fibroblast growth factor receptors, particularly FGFR3, in cancer has been illuminated. Dysregulation of oncogenes by translocation to the immunoglobulin heavy chain (IgH) locus on 14q32 is a seminal event in the pathogenesis of B-cell tumors. In multiple myeloma, translocations to the IgH locus occur in 20 to 60% of cases. For most translocations, the partner chromosome is unknown; for the others, a diverse array of chromosomal partners have been identified, with 11q13, the only chromosome that is frequently involved. Bergsagel et al. identified illegitimate switch recombination fragments (defined as containing sequences from only 1 switch region) as potential markers of translocation events into IgH switch regions in 15 of 21 myeloma cell lines, including 7 of 8 karyotyped lines that had no detectable 14q32 translocation. These translocation breakpoints involved 6 chromosomal loci: 4p16.3; 6; 8q24.13; 11q13.3; 16q23.1; and 21q22.1 (Bergsagel et al., Proc. Nat. Acad. Sci. 93:13931-13936, 1996). Chesi et al. (Nature Genet. 16:260-264 1997) found the karyotypically silent translocation t(4;14)(p16.3;q32.3) in 5 myeloma cells lines and in at least 3 of 10 primary tumors associated with multiple myeloma to exhibit increased expression and activation of mutations of FGFR3. The chromosome-4 breakpoints were clustered in a 70-kb region centromeric to FGFR3, which was thought to be the dysregulated oncogene. Two lines and 1 primary tumor with this translocation selectively expressed an FGFR3 allele containing activating mutations identified previously in thanatophoric dwarfism: tyr373 to cys, lys650 to glu, and lys650 to met. For K650E, the constitutive activation of FGFR3 in the absence of ligand had been proved by transfection experiments. Chesi et al. (1997) proposed that after the t(4;14) translocation, somatic mutation during tumor progression frequently generates an FGFR3 protein that is active in the absence of ligand.
Rasmussen, T et al. cited a frequency of 3 to 24% for the t(4;14) translocation in multiple myeloma (Rasmussen, T et al., Br. J. Haematol. 117:626-628, 2002). The translocation was observed at a significantly lower frequency in patients with monoclonal gammopathy of undetermined significance (MGUS), suggesting a role in the transition from MGUS to multiple myeloma. The t(4;14) translocation affects 2 potential oncogenes: FGFR3 and multiple myeloma set domain (MMSET). Rasmussen et al. (2002) investigated the frequency of FGFR3 dysregulation and its prognostic value in multiple myeloma. In 16 of 110 (14.5%) multiple myeloma bone marrow samples, they found dysregulated FGFR3 expression.
In addition, further evidence has been presented indicating an oncogenic role for FGFR3 in carcinomas (Cappellen, D. et al., (Letter) Nature Genet. 23:18-20, 1999). Cappellen et al. found expression of a constitutively activated FGFR3 in a large proportion of 2 common epithelial cancers, bladder and cervix. FGFR3 appeared to be the most frequently mutated oncogene in bladder cancer, being mutated in more than 30% of cases. FGFR3 seems to mediate opposite signals, acting as a negative regulator of growth in bone and as an oncogene in several tumor types. All FGFR3 missense somatic mutations identified in these cancers were identical to the germinal activating mutations that cause thanatophoric dysplasia (the authors noted that in 2 mutations, this equivalency occurred because the FGFR3b isoform expressed in epithelial cells contains 2 more amino acids than the FGFR3c isoform expressed in bone). Of the FGFR3 alterations in epithelial tumors, the S249C mutation was the most common, affecting 5 of 9 bladder cancers and 3 of 3 cervical cancers.
Evidence has also been presented indicating that activated FGFR3 is targeted for lysosomal degradation by c-Cbl-mediated ubiquitination, and that activating mutations found in patients with achondroplasia and related chondrodysplasias disturb this process, leading to recycling of activated receptors and amplification of FGFR3 signals (Cho et al., Proc. Nat. Acad. Sci. 101:609-614, 2004). Cho et al. suggested that this mechanism contributes to the molecular pathogenesis of achondroplasia and represents a potential target for therapeutic intervention. The lysosomal targeting defect is additive to other mechanisms proposed to explain the pathogenesis of achondroplasia.
Other results indicate that FGFR2 and FGFR3 are significant factors in tumorigenesis (Jang J H et al., “Mutations in fibroblast growth factor receptor 2 and fibroblast growth factor receptor 3 genes associated with human gastric and colorectal cancers” Cancer Res. 61(9):354 1-3, 2001). Due to their role in multiple myeloma, bladder cancer, and tumorigenesis, development of inhibitors of fibroblast growth factor receptor kinases, particularly inhibitors of FGFR2 and FGFR3, will play an import role in the treatment of cancers.
c-Kit is another receptor tyrosine kinase belonging to PDGF Receptor family and is normally expressed in hematopoietic progenitor, mast and germ cells. C-kit expression has been implicated in a number of cancers including mast cell leukemia, germ cell tumors, small-cell lung carcinoma, gastrointestinal stromal tumors, acute myelogenous leukemia (AML), erythroleukemia, neuroblastoma, melanoma, ovarian carcinoma, breast carcinoma (Heinrich, M. C. et al; J. Clin. One. 20, 6 1692-1703, 2002 (review article); Smolich, B. D. et al., Blood, 97, 5; 1413-1421).
Overexpression of CSF-1R, the receptor for colony stimulating factor-1 (CSF-1) has been implicated in a number of human carcinomas, including carcinomas of the breast, ovary, endometrium, lung, kidney, pancreas and prostate (Sapi, E., Exp. Biol. Med 229:1-11, 2004). CSF-1R is tyrosine kinase receptor which, when activated by its ligand CSF-1, triggers signal transduction pathways controlling cell proliferation and differentiation. CSF-1R is expressed in the mammary gland during pregnancy and lactation. Abnormal CSF-1R expression has been correlated with 58% of all breast cancers, and with 85% of invasive breast carcinoma (see Sapi, supra).
A continuing need exists for compounds that inhibit the proliferation of capillaries, inhibit the growth of tumors, treat cancer, modulate cell cycle arrest, and/or inhibit molecules such as one or more of Ras, Raf, mutant B-Raf, VEGFR2 (KDR, Flk-1), FGFR2/3, c-Kit, PDGFRβ, CSF-1R, and pharmaceutical formulations and medicaments that contain such compounds. A need also exists for methods of administering such compounds, pharmaceutical formulations, and medicaments to patients or subjects in need thereof.